Project Summary/Abstract Existing medicines for tuberculosis are losing ground to drug resistance. There is intense need to develop new regimens that include drugs active against Mycobacterium tuberculosis (Mtb) cells in the slowly- or non-replicating states (for simplicity, ?NR? states) associated with phenotypic tolerance to most TB drugs. Our long-term goal is to develop anti-Mtb drugs that kill NR Mtb populations to complement drugs that kill replicating Mtb populations. Mtb relies on specific enzymatic pathways to survive the host stresses that make it NR. Among these is the prokaryotic ubiquitin-like protein (Pup)-proteasome system discovered over several years beginning in 2003. Although the Mtb proteasome is dispensable under standard growth conditions, it has been validated as a drug target both genetically and pharmacologically: the knock-out strain dies in the chronic phase of disease in mice, and we introduced several classes of Mtb proteasome inhibitors that kill NO-stressed or starved Mtb in vitro. These included the first inhibitors selective (>1000-fold) for the proteasome of a pathogen and not its host, and the first agents to kill bacteria by inhibiting protein breakdown rather than protein synthesis. Heretofore there have been only five broad classes of antibiotic targets: inhibition of synthesis of nucleic acids, proteins, cell walls and folate, and disruption of membranes. Our work has validated a fifth class: inhibition of a protein degradation pathway, and led to the discovery by others of pathogen-selective proteasome inhibitors that kill the protozoal agents of malaria, Chagas disease and Leishmaniasis. In this application, we will advance our newly discovered novel class of peptidomimetics. We demonstrate that these inhibitors are highly potent and species selective Mtb proteasome inhibitors. We propose to conduct lead optimization to achieve oral bioavailability with safety for future in vivo efficacy studies in mouse model of Mtb infection.